Organic selenium-rich microbial inoculum as well as preparation method and application thereof

Abstract

The invention provides an organic selenium-rich microbial inoculum as well as a preparation method and application thereof, and relates to the technical field of microbial inoculum. The organic selenium-rich microbial inoculum comprises selenium-substituted amino acid; based on 100 parts by mass of organic selenium, the content of the seleno-amino acid in the organic selenium-rich microbial inoculum is not less than 5%. The invention further provides a preparation method and application of the organic selenium-rich microbial inoculum. The organic selenium-rich microbial inoculum provided by the invention overcomes the defect that the conversion rate is low when inorganic selenium is converted into organic selenium by microorganisms in the prior art.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to an organic selenium-rich bacterial agent and a preparation method and application thereof. Background Art

[0002] Selenium is an essential trace element for humans and animals. Current studies have shown that a daily intake of 50 micrograms of selenium per person can keep people healthy and long-lived, and supplementing organic selenium through food is considered the safest and most convenient means. In addition, in the absorption and conversion of selenium required by the human body, it is currently found that amino acid organic selenium, which is derived from traditional organic selenium such as protein selenium and polysaccharide selenium, is more easily absorbed and converted in the human body.

[0003] In the prior art, the supplement of organic selenium is usually provided by plant organic selenium, and the organic selenium in plants is usually obtained by soil absorption and conversion. Based on this, selenium-rich fertilizers have become a research hotspot. However, the utilization rate of inorganic selenium in crops is low, and it is difficult to meet the demand for selenium-rich agricultural products. Therefore, selenium-rich fertilizers containing organic selenium have become a new research hotspot. Summary of the invention

[0004] One of the purposes of the present application is to provide an organic selenium-rich bacterial agent and a preparation method and application thereof, wherein the organic selenium-rich bacterial agent is more easily absorbed by plants.

[0005] In a first aspect, the present application discloses an organic selenium-rich bacterial agent, comprising organic selenium obtained by converting inorganic selenium, wherein the content of organic selenium is not less than 80% by mass fraction of the total selenium;

[0006] The organic selenium includes selenoamino acids, selenopolypeptides, selenoproteins and selenopolysaccharides obtained by conversion of inorganic selenium; based on 100 parts by mass of organic selenium, the organic selenium includes 30 to 35 parts of selenopolypeptides, 18 to 25 parts of selenoproteins, 30 to 45 parts of selenopolysaccharides and 5 to 15 parts of selenoamino acids; the content of inorganic selenium in the organic selenium bacterial agent is not higher than 20% and not lower than 2% in terms of the mass fraction of the total selenium.

[0007] Furthermore, in some embodiments of the present application, the selenoamino acid includes one or more of selenomethionine and selenocysteine.

[0008] Furthermore, in some embodiments of the present application, the organic-selenium-rich bacterial agent also includes calcium protein and phospholipids, and the concentration of the calcium protein in the organic-selenium-rich bacterial agent is not less than 0.001 g / L; the concentration of the phospholipid in the organic-selenium-rich bacterial agent is not less than 0.001 g / L.

[0009] Furthermore, in some embodiments of the present application, the organic selenium-rich bacterial agent is obtained by fermenting an inorganic selenium solution with a composite photosynthetic bacterial community; wherein the composite photosynthetic bacterial community includes Rhodopseudomonas palustris, Rhodospirillum rubrum, Nitrogen-fixing Rhodobacter, Rhodobacter capsulatum, and Rhodopseudomonas thiophila.

[0010] Furthermore, in some embodiments of the present application, the total viable count of the photosynthetic bacteria complex in the organic selenium-rich bacterial agent is not less than 200 million / mL.

[0011] In a second aspect, the present application also provides a method for preparing an organic selenium-rich bacterial agent, which comprises inoculating a composite flora of photosynthetic bacteria into a culture solution, adding an inorganic selenium salt, and fermenting and culturing the solution;

[0012] The photosynthetic bacteria complex includes Rhodopseudomonas palustris, Rhodospirillum rubrum, Nitrogen-fixing Rhodobacter, Rhodobacter capsulatum, and Rhodopseudomonas thiophila.

[0013] Further, in some embodiments of the present application, the culture solution includes the following components by mass: 78-92 parts of magnesium sulfate, 720-880 parts of anhydrous sodium acetate, 32-38 parts of anhydrous calcium chloride, 450-550 parts of urea, 440-520 parts of dipotassium hydrogen phosphate, 280-340 parts of potassium dihydrogen phosphate, 95-115 parts of yeast extract, 22-28 parts of protein extract, 0.13-0.17 parts of niacin, 3.5-4.5 parts of boric acid and 570,000-700,000 parts of water;

[0014] The concentration of the inorganic selenium salt formed by adding the inorganic selenium salt into the culture solution is 0.63-0.77 g / L.

[0015] Furthermore, in some embodiments of the present application, the pH value of the culture solution is 7.7 to 7.8; and / or

[0016] The fermentation culture conditions are: temperature 30-38, stirring speed 18-22r / min, stirring for 2.5-3.5min every 2.5-3.5 hours, and fermentation time 500-700h.

[0017] Furthermore, in some embodiments of the present application, after the photosynthetic bacteria composite flora is inoculated into the culture solution, it is cultured for 18 to 22 hours, and then the inorganic selenium salt is added.

[0018] In a third aspect, the present application also provides the use of the organic-selenium-rich bacterial agent prepared by the preparation method of the organic-selenium-rich bacterial agent described in the first aspect or the organic-selenium-rich bacterial agent described in the second aspect in crop planting.

[0019] Beneficial effects of this application:

[0020] The present application provides an organic selenium-rich bacterial agent, wherein the organic selenium of the bacterial agent contains at least 5% of water-soluble selenoamino acids, which are not only beneficial to the plant's absorption of organic selenium in the soil or sprayed on other parts of the plant; but also because the selenoamino acids have better stability, are easier to combine with other organic matter in the plant, and are more quickly transported, they are beneficial to achieving selenium-enrichment in the rhizomes, fruits, stems and leaves of the plant, making it more widely used in selenium-rich plants; in addition, the selenoamino acids entering the plant body can further promote the plant to convert inorganic selenium into selenoproteins, and can also promote the plant metabolism to generate other selenoamino acids, enrich the types of selenoamino acids provided by the plant, and improve the value of the plant and the human body's absorption of selenium in the plant.

[0021] The present application provides a method for preparing an organic selenium-rich bacterial agent, which adopts a composite bacterial community formed by nitrogen-fixing red bacteria, capsule red bacteria, palustris red bacteria, crimson red bacteria, and sulfur-loving red bacteria. The composite utilizes nitrogen-fixing red bacteria and capsule red bacteria, which are microorganisms with good performance of converting inorganic selenium into organic selenium, and adds sulfur-loving red bacteria that can convert selenium after a stable reproduction period, so as to improve the conversion performance of the bacterial community on inorganic selenium. At the same time, palustris red bacteria and crimson red bacteria are also added to the bacterial community, and the inorganic selenium is converted into a variety of organic selenium, such as selenoproteins, selenopolypeptides, selenopolysaccharides and selenoamino acids, by utilizing the synergistic effect of the composite bacterial community formed by multiple bacteria. Not only the conversion rate of inorganic selenium is improved, but also the richness of organic selenium in the organic selenium bacterial agent obtained by using the composite bacterial community can be improved, the absorption effect of crops on organic selenium can be optimized, and the application of organic selenium in promoting the growth of crops can be expanded.

[0022] The present application provides an organic selenium-rich bacterial agent, the conversion rate of which for inorganic selenium can reach 80%, or even 95%. The organic selenium-rich bacterial agent provided by the present application contains a rich variety of organic selenium and can be used as a fertilizer for crops. It not only significantly increases production and has high product quality, but also significantly increases the selenium content in the products and the selenium content in various parts of the crops, which is conducive to the widespread use of the bacterial agent on crops. DETAILED DESCRIPTION

[0023] In order to better explain the present invention, the following description will be given in detail with reference to the embodiments of the present invention, and the main contents of the present invention will be further clarified in conjunction with specific examples. However, the contents of the present invention are not limited to the following examples.

[0024] In the prior art, most of the research on organic selenium-rich fertilizers or organic selenium-rich microbial agents added to fertilizers focuses on the general content of organic selenium in fertilizers or microbial agents, while most of the research on plant absorption of fertilizers or microbial agents still focuses on increasing the organic selenium content in the soil to increase the absorption of selenium by plants. However, there are few studies on the types of organic selenium in fertilizers or microbial agents and the absorption effects of organic selenium by plants. The absorption of selenium by plants is the key to increasing the selenium content in selenium-rich plants.

[0025] In order to solve this technical problem, the inventors proposed an organic selenium-rich bacterial agent, including organic selenium obtained by converting inorganic selenium, wherein the content of organic selenium is not less than 80% by mass fraction of the total selenium;

[0026] The organic selenium includes selenoamino acids, selenopolypeptides, selenoproteins and selenopolysaccharides obtained by conversion of inorganic selenium; based on 100 parts by mass of organic selenium, the organic selenium includes 30 to 35 parts of selenopolypeptides, 18 to 25 parts of selenoproteins, 30 to 45 parts of selenopolysaccharides and 5 to 15 parts of selenoamino acids; the content of inorganic selenium in the organic selenium bacterial agent is not higher than 20% and not lower than 2% in terms of the mass fraction of the total selenium.

[0027] The absorption of selenium by plants is not only directly related to the abundance and deficiency of selenium in the soil, but also to the form of selenium. Selenium in different forms has different chemical and biological properties, and its effectiveness to plants varies greatly. The absorption of water-soluble selenium, especially water-soluble organic selenium, is faster than that of other forms of selenium. For example, wheat seedlings and rapeseed seedlings absorb selenomethionine more than 100 times faster than selenate. Moreover, water-soluble organic selenium can further promote plants to convert inorganic selenium in soil or fertilizer into organic selenium, and convert existing organic selenium into other organic selenium, thereby increasing the content of organic selenium in plants and the richness of organic selenium species. For the human body, organic selenium is less toxic and more suitable for the human body, especially selenoamino acids, because they are water-soluble selenium-containing compounds, which are not only less toxic, but also highly active, easier for the human body to absorb, and have a higher utilization rate after absorption. Therefore, increasing the content of organic selenium in plants, the types of organic selenium, especially the content and types of selenoamino acids, is conducive to the absorption and utilization of selenium by the human body when eating selenium-rich crops. In addition, different parts of crops have different requirements for selenium absorption and different forms of selenium have different transport rates in crops. Therefore, the utilization rate of microbial agents with the same selenium content is not the same for different crops. Generally speaking, selenium is more easily enriched in the root system of plants, and its selenium enrichment is higher. Therefore, for crops whose stems, leaves, and fruits are edible, the utilization rate of selenium in the plants is lower, and it is necessary to promote the transport of selenium in the root system of plants to edible or more edible stems, leaves, or fruits.

[0028] The microbial agent used in the present application includes organic selenium and inorganic selenium, wherein the organic selenium includes at least 5% selenoamino acid, which has good water solubility and is more conducive to entering the soil and spraying on stems and leaves through a solution, promoting the rapid absorption of selenium by plants, and does not require plants to consume a large amount of energy. In addition, the selenoamino acid absorbed into the plant is conducive to promoting the absorption, conversion and transportation of other organic selenium and even inorganic selenium, making the types of organic selenium in the plant more abundant, and can migrate from the root system of the plant to the stems and leaves and fruit parts of the plant, promoting the increase of the content of organic selenium in the stems and leaves and fruits of the plant, and is particularly suitable for crops that mainly eat fruits and stems and leaves. At the same time, it is also conducive to the harvest of fruits and stems and leaves of the plant, reducing the selenium enrichment in the root system of the plant after the root system absorbs a large amount of selenium in the soil, and producing obvious selenium absorption inhibition.

[0029] The organic selenium in the organic selenium-rich bacterial agent also includes selenium polypeptides, selenoproteins and selenium polysaccharides;

[0030] Taking 100 parts by mass of organic selenium, the organic selenium comprises 30 to 35 parts of selenium polypeptide, 18 to 25 parts of selenoprotein, 30 to 45 parts of selenium polysaccharide and 5 to 15 parts of selenoamino acid.

[0031] The selenopolysaccharide may be one or more of selenized chitosan, selenate polysaccharide, selenized heteropolysaccharide and other selenium-containing polysaccharides. Preferably, the selenopolysaccharide is at least two, and the selenopolypeptide and selenoprotein also include at least two, respectively.

[0032] In some embodiments, the organic selenium-rich bacterial agent further comprises calcium protein and phospholipids, wherein the concentration of calcium protein in the organic selenium-rich bacterial agent is not less than 0.001 g / L; the concentration of phospholipids in the organic selenium-rich bacterial agent is not less than 0.001 g / L. Preferably, the concentration of phospholipids in the organic selenium-rich bacterial agent is not less than 0.5 g / L, which is beneficial to promote the absorption of selenium and is converted into selenium phospholipids that have a significant effect on preventing overweight and obesity.

[0033] It should be noted that the concentrations of calcium protein and phospholipid in the organic selenium-rich bacterial agent are measured by drying the bacterial agent to a water content of no more than 70%.

[0034] The selenoamino acid comprises one or more of selenomethionine and selenocysteine, preferably both selenomethionine and selenocysteine. More preferably, the content of selenomethionine is not less than 1% and the content of selenocysteine ​​is not less than 1% based on 100 parts by mass of organic selenium.

[0035] The content of organic selenium in the organic selenium-rich bacterial agent is not less than 80% by mass fraction of the total selenium; the content of inorganic selenium in the organic selenium bacterial agent is not higher than 20% and not lower than 2% by mass fraction of the total selenium. Preferably, the content of organic selenium is not less than 85% by mass fraction of the total selenium; the content of inorganic selenium in the organic selenium bacterial agent is not higher than 15% and not lower than 2% by mass fraction of the total selenium. Further preferably, the content of organic selenium is not less than 90% by mass fraction of the total selenium; the content of inorganic selenium in the organic selenium bacterial agent is not higher than 10% and not lower than 2% by mass fraction of the total selenium.

[0036] It should be noted that in the present application, a small amount of water-soluble inorganic selenium contained in an organic selenium-rich bacterial agent may be better than one without a small amount of water-soluble inorganic selenium, especially selenite. It can adjust the pH value of the soil, stabilize it near neutral soil, consume excessive oxygen in the soil, and promote the absorption of selenium by plant roots. This is because in a neutral or even slightly alkaline soil environment, the plant roots absorb selenium more rapidly. However, the content of water-soluble inorganic selenium should not be too high. Excessive water-soluble inorganic selenium has a certain toxic effect on soil and plants, so it is very necessary to control the water-soluble selenium content in the bacterial agent.

[0037] In some embodiments, the organic selenium-rich bacterial agent is obtained by fermenting an inorganic selenium solution with a composite photosynthetic bacterial community; wherein the composite photosynthetic bacterial community includes Rhodopseudomonas palustris, which is numbered bio-52488 in China Microbial Species, Rhodospirillum rubrum, which is numbered bio-53129 in China Microbial Species, Nitrogen-fixing Rhodobacter, which is numbered bio-02221 in China Microbial Species, Rhodobacter capsulatum, which is numbered bio-52491 in China Microbial Species, and Rhodopseudomonas thiophila, which is numbered bio-67435 in China Microbial Species.

[0038] In the present application, Rhodopseudomonas palustris, Rhodospirillum crimsonii, Rhodobacter azotobacter, Rhodobacter capsulatum, and Rhodopseudomonas sulfophilus can be purchased from the market, such as from ATCC (American Type Culture Collection); or they can be obtained by self-cultivation using commercially purchased strains. For example, purchased strains of Rhodopseudomonas palustris (bio-52488, number: DSM5859), Rhodospirillum crimsonii, Rhodobacter azotobacter (bio-02221), Rhodobacter capsulatum (bio-52491, number: ATCC11166), and Rhodopseudomonas sulfophilus are used, and the strains are used to culture a separate bacterial community, and then the bacterial communities are inoculated together in a culture medium to culture to obtain a photosynthetic bacterial composite community.

[0039] Among them, the culture medium used when culturing Rhodopseudomonas palustris, Rhodospirillum crimsonii, Rhodobacter azotobacter, Rhodobacter capsulatum, and Rhodopseudomonas sulfidophilus alone can be a conventional commercially available formula. For example, the culture medium formulas in the culture process of Rhodopseudomonas palustris and Rhodobacter capsulatum can be as shown in Table 1:

[0040] Table 1

[0041] Element Mass parts (parts) Dipotassium hydrogen phosphate 1.0 Magnesium sulfate 0.5 Yeast Extract 10.0 Distilled water 1000

[0042] The culture conditions are 30 anaerobic environment, pH value of 7.0-7.2, and culture under 40W tungsten filament light for 5-7 days. Before inoculating the bacteria, the culture environment is sterilized at 121 high pressure for 15 minutes.

[0043] Exemplarily, the culture medium formula during the cultivation of nitrogen-fixing red bacteria can be as shown in Table 2:

[0044] Table 2

[0045] Element Mass parts (parts) Element Mass parts (parts) Potassium dihydrogen phosphate 1.0 Magnesium chloride 0.5 Sodium acetate 1.0 Sodium bicarbonate 3.0 Sodium chloride 1.0 Ammonium chloride 1.0 Distilled water 1000 Sodium succinate 1.0 Yeast Extract 0.50 Peptone 0.5 Calcium chloride 0.1

[0046] Trace element solution and vitamin solution are also added to the formula, and the volume ratio of the added amount to distilled water is 1:1000 respectively. The pH value of the formula is 6.8.

[0047] The formula of the trace element solution and the vitamin solution can be shown in Table 3:

[0048] Table 3

[0049]

[0050] Exemplarily, the culture medium formula during the cultivation of Rhodospirilla rubrum can be as shown in Table 4:

[0051] Table 4

[0052]

[0053]

[0054] The formula also includes a vitamin B12 component, which is an aqueous solution with a concentration of 0.1 mg / mL, and the volume ratio of the vitamin B12 component to distilled water is 0.4:1050. The formula also includes a trace element solution, and the volume ratio of the trace element solution to distilled water is 1:1000. The pH value of the formula is 6.8.

[0055] The formula of the trace element solution can be shown in Table 5:

[0056] Table 5

[0057] Element Mass parts (parts) Cupric chloride dihydrate 0.01 Cobalt chloride hexahydrate 0.20 Distilled water 1000 Nickel chloride dihydrate 0.02 Zinc sulfate heptahydrate 0.1 Manganese chloride tetrahydrate 0.03 Sodium molybdate dihydrate 0.03 Boric acid 0.3

[0058] Exemplarily, the medium formula in the culturing process of Rhodopseudomonas thiophilus can be as shown in Table 6:

[0059] Table 6

[0060] Element Mass parts (parts) Element Mass parts (parts) Potassium dihydrogen phosphate 1.0 Magnesium chloride hexahydrate 0.5 Sodium acetate 1.0 Sodium bicarbonate 3.0 Sodium chloride 30.0 Ammonium chloride 1.0 Distilled water 1000 Sodium sulfate 0.7 Yeast extract 0.1 Sodium Ascorbate 0.5 Calcium chloride dihydrate 0.1 Sodium sulfide 0.156

[0061] The formula also includes trace element solution and vitamin solution, the volume ratio of the added amount to distilled water is 1:1000, and the pH value of the formula is 6.9-7.0.

[0062] The formula of the trace element solution and the vitamin solution can be shown in Table 7:

[0063] Table 7

[0064]

[0065] It should be noted that the culture medium used separately in the above-mentioned bacterial culture process is only an exemplary culture medium that can be used, and is not a limitation on the culture process of each bacteria used in this application. Those skilled in the art can make adjustments within a certain range to enable each bacteria to grow normally during the culture process.

[0066] Furthermore, in the actual culture process, the above-mentioned bacteria are cultured separately, and several generations can be cultured in the culture process, such as culturing each bacteria to the third generation, and then the bacteria are inoculated together for mixed culture. During the inoculation process, the inoculation amount of each bacteria is as similar as possible, and then the culture medium in the mixed culture process is used to culture a composite bacterial community. The culture medium used in the mixed culture process may include the following components: 78-92 parts of magnesium sulfate, 720-880 parts of anhydrous sodium acetate, 32-38 parts of anhydrous calcium chloride, 450-550 parts of urea, 440-520 parts of dipotassium hydrogen phosphate, 280-340 parts of potassium dihydrogen phosphate, 95-115 parts of yeast extract, 22-28 parts of protein extract, 0.13-0.17 parts of niacin, 3.5-4.5 parts of boric acid and 570,000-700,000 parts of water. For example, the composition of the culture medium may be: 85 parts of magnesium sulfate, 800 parts of anhydrous sodium acetate, 35 parts of anhydrous calcium chloride, 500 parts of urea, 480 parts of dipotassium hydrogen phosphate, 313 parts of potassium dihydrogen phosphate, 105 parts of yeast extract, 25 parts of protein extract, 0.15 parts of niacin, 4 parts of boric acid and 630,000 parts of water. The pH value of the culture medium is 7.7-7.8, and the culture conditions are sterilized by ozone, cultured in an anaerobic environment of not less than 30, and under light.

[0067] In some embodiments, the composite photosynthetic bacteria community can grow normally in a fermentation broth with an inorganic selenium concentration of not less than 336 mg / kg; preferably, the composite photosynthetic bacteria community can grow normally in a fermentation broth with an inorganic selenium concentration of not less than 480 mg / kg, and the conversion rate of the inorganic selenium to organic selenium is not less than 80%. The inorganic selenium can be an inorganic selenium salt, preferably a selenite, such as sodium selenite.

[0068] Furthermore, in some embodiments of the present application, the photosynthetic bacteria composite flora includes no more than 0.3% of other bacteria to limit the influence of other bacteria on the growth of each bacteria in the composite flora.

[0069] In a second aspect, the present application also provides the use of the composite photosynthetic bacteria community described in the first aspect in an organic selenium-rich bacterial agent for converting inorganic selenium into organic selenium.

[0070] In this application, the inventor provides a specific application of a composite photosynthetic bacteria community in an organic-selenium-rich bacterial agent: an organic-selenium-rich bacterial agent and a preparation method thereof, wherein the organic-selenium-rich bacterial agent comprises the composite photosynthetic bacteria community described in the first aspect; the number of viable bacteria of the composite photosynthetic bacteria community in the organic-selenium-rich bacterial agent is not less than 200 million / mL, and the content of organic selenium in the organic-selenium-rich bacterial agent is not less than 560 mg / L.

[0071] Since the conversion rate of inorganic selenium by the photosynthetic bacterial composite flora used in the present application can reach more than 80%, the content of inorganic selenium in the obtained organic selenium-rich bacterial agent is lower than the content of organic selenium, thereby improving the utilization rate of selenium in the bacterial agent by the plants and reducing the amount of the bacterial agent used.

[0072] More preferably, the conversion rate of inorganic selenium by the composite photosynthetic bacteria used in the present application can reach more than 90%, so that the content of inorganic selenium in the organic selenium-rich bacterial agent is less than 10%.

[0073] In addition, the organic selenium contained in the organic selenium-rich bacterial agent obtained by converting the composite flora of photosynthetic bacteria used in the present application includes a variety of organic selenium such as selenoprotein, selenopolypeptide, selenopolysaccharide, selenoamino acid, etc., and its organic selenium is rich in variety, which is more conducive to the utilization of selenium in the bacterial agent by plants. Among them, the proportion of selenoamino acid in organic selenium is not less than 5% by mass, including at least 1% of selenomethionine and at least 1% of selenocysteine. Among them, more preferably, the content of selenomethionine is not less than 2%, and the content of selenocysteine ​​is not less than 2%.

[0074] Preferably, selenoprotein, selenopolypeptide and selenohybrid polysaccharide are the main components of organic selenium in the formed organic selenium-rich bacterial agent, and their mass fraction can reach more than 20% of the organic selenium. At the same time, in the present application, the content of each main organic selenium is relatively uniform, and the obtained organic selenium-rich bacterial agent is better.

[0075] The present application also provides a method for preparing the organic selenium-rich bacterial agent, which comprises: inoculating the composite photosynthetic bacteria community described in the first aspect into a culture solution, adding an inorganic selenium salt, and fermenting and culturing the solution.

[0076] In some embodiments, the culture solution includes the following components by mass: 78-92 parts of magnesium sulfate, 720-880 parts of anhydrous sodium acetate, 32-38 parts of anhydrous calcium chloride, 450-550 parts of urea, 440-520 parts of dipotassium hydrogen phosphate, 280-340 parts of potassium dihydrogen phosphate, 95-115 parts of yeast extract, 22-28 parts of protein extract, 0.13-0.17 parts of niacin, 3.5-4.5 parts of boric acid, and 570,000-700,000 parts of water;

[0077] The concentration of the inorganic selenium salt formed by adding the inorganic selenium salt into the culture solution is 0.63-0.77 g / L.

[0078] Exemplarily, the culture solution includes the following ingredients in parts by mass: 85 parts of magnesium sulfate, 800 parts of anhydrous sodium acetate, 35 parts of anhydrous calcium chloride, 500 parts of urea, 480 parts of dipotassium hydrogen phosphate, 313 parts of potassium dihydrogen phosphate, 105 parts of yeast extract, 25 parts of protein extract, 0.15 parts of niacin, 4 parts of boric acid and 630,000 parts of water.

[0079] Exemplarily, the concentration of the inorganic selenium salt formed by adding the inorganic selenium salt to the culture solution is 0.7 g / 10L.

[0080] In some embodiments, the pH value of the culture solution is 7.7-7.8; and / or

[0081] The fermentation culture conditions are: temperature 30-38, pressure (normal pressure), stirring speed 18-22r / min, stirring for 2.5-3.5min every 2.5-3.5 hours, and fermentation time 500-700h.

[0082] Exemplarily, the fermentation culture conditions are: under anaerobic conditions, temperature 30-38, pressure atmospheric pressure Pa, stirring speed 20r / min, stirring for 3 minutes every 3 hours, and fermentation time 600h.

[0083] In some embodiments, after the photosynthetic bacteria complex is inoculated in the culture medium, it is cultured for 18 to 22 hours, and then inorganic selenium salt is added to allow the photosynthetic bacteria complex to adapt to the environment in the culture medium, which is more conducive to its stable and normal growth and conversion of inorganic selenium salt.

[0084] In some embodiments, the inorganic selenium salt is added to the culture medium only once.

[0085] In some embodiments, in order to extend the shelf life of the organic selenium-rich bacterial agent provided in the present application, a preservative can be added thereto so that it can be stored for a long time, or the concentrated freeze-dried powder can be obtained to facilitate its storage and transportation. Among them, the preservative can be selected from one or more of E-polylysine, sodium dehydroacetate, calcium propionate, sodium diacetate, sodium lactate, and nisin. The amount of preservative added can be adjusted according to the conventional techniques of those skilled in the art, such as, the amount of preservative added can be 0.2% of the fermentation broth in terms of mass fraction.

[0086] In a fifth aspect, the present application also provides the application of an organic selenium-rich bacterial agent in crop planting. It can be used as an organic selenium fertilizer in the process of crop planting, and is used by spraying or other fertilization methods in the process of crop planting. In some embodiments, the organic selenium-rich bacterial agent can be directly diluted before application. For example, the fermentation liquid obtained can be diluted 50 times and applied in crop planting to increase the content of organic selenium in the soil, thereby obtaining selenium-rich crops, thereby increasing the utilization rate of selenium in the soil, and reducing the damage to the soil caused by the application of inorganic selenium fertilizers.

[0087] The diluted microbial agent can be applied by spraying the diluted microbial agent intermittently on the leaves of crops in the growing period or the leaves of economic forests 1 to 3 times, and the interval between two adjacent sprayings can be 15-20 days.

[0088] The following is an explanation of several embodiments related to the present application, but it is not intended to limit the present application to these embodiments. The raw materials or reagents involved in the present application can be obtained commercially; the operations involved are conventional operations in the field unless otherwise specified. This application adopts GB5009.93-2010 (first method) to detect the total selenium content in the fermentation broth and adopts

[0089] The content of organic selenium in the fermentation broth is tested in the study of the determination method of organic selenium in selenium-enriched yeast health foods.

[0090] Example 1

[0091] The cultivation of the photosynthetic bacteria composite flora comprises the following steps:

[0092] (1) purchasing strains of Rhodopseudomonas palustris, Rhodospirillum crimsonii, Rhodobacter azotobacter, Rhodobacter capsulatum, and Rhodopseudomonas sulfidophilus, and culturing them respectively in the culture media recorded in Tables 1 to 7 to obtain the third generation, and isolating colonies of Rhodopseudomonas palustris, Rhodospirillum crimsonii, Rhodobacter azotobacter, Rhodobacter capsulatum, and Rhodopseudomonas sulfidophilus;

[0093] (2) inoculating the Rhodopseudomonas palustris, Rhodospirillum crimsonii, Rhodobacter azotobacter, Rhodobacter capsularis, and Rhodopseudomonas thiophilus obtained in step (1) into a culture medium of composite bacteria, wherein the formula of the culture medium is:

[0094] 1.2 g of magnesium sulfate, 8 g of anhydrous sodium acetate, 2.7 g of anhydrous calcium chloride, 2.7 g of urea, 5.5 g of dipotassium hydrogen phosphate, 3.7 g of potassium dihydrogen phosphate, 0.45 g of yeast extract, 0.01 g of niacin, 0.1 g of boric acid, and 10 L of ozone-sterilized water.

[0095] The mixed culture is used to obtain a mixed bacterial community of Rhodopseudomonas palustris, Rhodospirillum crimsonii, Rhodobacter azotobacter, Rhodobacter capsularis, and Rhodopseudomonas sulfidophilus with a total viable count of not less than 200 million / ml, thereby obtaining a photosynthetic bacteria composite bacterial community. The photosynthetic bacteria composite bacterial community is then expanded and cultured for later use.

[0096] (3) The cultured photosynthetic bacterial composite flora was inoculated into 70 L of a culture solution of a bacterial agent having a pH value in the range of 7.7 to 7.8, wherein the formula of the culture solution of the bacterial agent is:

[0097] 85g magnesium sulfate, 800g anhydrous sodium acetate, 35g anhydrous calcium chloride, 500g urea, 480g dipotassium hydrogen phosphate, 313g potassium dihydrogen phosphate, 105g yeast extract, 25g protein extract,

[0098] 0.15g niacin, 4g boric acid and 630L of ozone-sterilized water. The culture barrel used is also sterilized by ozone and deoxygenated by carbon dioxide or other gases, so that the sterilized culture barrel is in an anaerobic environment and is in a closed state.

[0099] After inoculating the cultured photosynthetic bacteria composite flora into the culture solution of the inoculant under sterile conditions, the photosynthetic bacteria composite flora was cultured under light at a temperature of 35 for 20 hours to allow the photosynthetic bacteria composite flora to adapt to the environment of the culture barrel, and then 490 g of sodium selenite was added at one time, and cultured for 600 hours at an intermittent stirring speed of 20 rad / min, wherein the stirring interval was 3 minutes every 3 hours, to obtain a inoculant, and the concentration of organic selenium in the inoculant was measured to be 192 mg / L, and the conversion rate of inorganic selenium was 90.6%.

[0100] Example 2

[0101] The cultivation of the photosynthetic bacteria composite flora comprises the following steps:

[0102] (1) purchasing strains of Rhodopseudomonas palustris, Rhodospirillum crimsonii, Rhodobacter azotobacter, Rhodobacter capsulatum, and Rhodopseudomonas sulfidophilus, and culturing them respectively in the culture media recorded in Tables 1 to 7 to obtain the third generation, and isolating colonies of Rhodopseudomonas palustris, Rhodospirillum crimsonii, Rhodobacter azotobacter, Rhodobacter capsulatum, and Rhodopseudomonas sulfidophilus;

[0103] (2) inoculating the Rhodopseudomonas palustris, Rhodospirillum crimsonii, Rhodobacter azotobacter, Rhodobacter capsularis, and Rhodopseudomonas thiophilus obtained in step (1) into a culture medium of composite bacteria, wherein the formula of the culture medium is:

[0104] 3 g of magnesium sulfate, 21 g of anhydrous sodium acetate, 4.5 g of anhydrous calcium chloride, 6 g of urea, 12 g of dipotassium hydrogen phosphate, 8 g of potassium dihydrogen phosphate, 1.5 g of yeast extract, 0.01 g of niacin, 0.1 g of boric acid, and 10 L of ozone-sterilized water.

[0105] The mixed bacterial community of Rhodopseudomonas palustris, Rhodospirillum crimsonii, Rhodobacter azotobacter, Rhodobacter capsularis, and Rhodopseudomonas sulfophilus with a total viable count of not less than 200 million / ml can be obtained by mixed culture, and the composite bacterial community of photosynthetic bacteria can then be obtained. The composite bacterial community of photosynthetic bacteria is then expanded and cultured for later use. (3) The composite bacterial community of photosynthetic bacteria obtained by culture is inoculated into 700 L of a culture solution of a bacterial agent with a pH value in the range of 7.7 to 7.8, wherein the formula of the culture solution of the bacterial agent is:

[0106] 100g magnesium sulfate, 850g anhydrous sodium acetate, 35g anhydrous calcium chloride, 364g urea, 550g dipotassium hydrogen phosphate, 350g potassium dihydrogen phosphate, 100g yeast extract, 0.7g niacin, 7g boric acid and 630L water sterilized by strong ultraviolet light. The culture barrel used is also sterilized by ozone, and the sterilized culture barrel is in an anaerobic environment and in a closed state through carbon dioxide or other deoxygenation.

[0107] After inoculating the cultured photosynthetic bacteria composite flora into the culture solution of the inoculant under sterile conditions, the photosynthetic bacteria composite flora was cultured under light at a temperature of 37 for 20 hours to allow the photosynthetic bacteria composite flora to adapt to the environment of the culture barrel, and then 490g of sodium selenite was added at one time, and the inoculant was cultured for 720 hours under intermittent stirring speed, wherein the stirring interval was 3 minutes every 2 hours to obtain the inoculant. The concentration of organic selenium in the inoculant was measured to be 186.5mg / L, and the conversion rate of inorganic selenium was 88%.

[0108] Example 3

[0109] The cultivation of the photosynthetic bacteria composite flora comprises the following steps:

[0110] (1) purchasing strains of Rhodopseudomonas palustris, Rhodospirillum crimsonii, Rhodobacter azotobacter, Rhodobacter capsulatum, and Rhodopseudomonas sulfidophilus, and culturing them respectively in the culture media recorded in Tables 1 to 7 to obtain the third generation, and isolating colonies of Rhodopseudomonas palustris, Rhodospirillum crimsonii, Rhodobacter azotobacter, Rhodobacter capsulatum, and Rhodopseudomonas sulfidophilus;

[0111] (2) inoculating the Rhodopseudomonas palustris, Rhodospirillum crimsonii, Rhodobacter azotobacter, Rhodobacter capsularis, and Rhodopseudomonas thiophilus obtained in step (1) into a culture medium of composite bacteria, wherein the formula of the culture medium is:

[0112] 2.5 g of magnesium sulfate, 19 g of anhydrous sodium acetate, 5.5 g of anhydrous calcium chloride, 5.6 g of urea, 11.69 g of dipotassium hydrogen phosphate, 7.56 g of potassium dihydrogen phosphate, 1 g of yeast extract, 0.01 g of niacin, 0.1 g of boric acid, and 10 L of ozone-sterilized water.

[0113] The mixed bacterial flora of Rhodopseudomonas palustris, Rhodospirillum crimsonii, Rhodobacter azotobacter, Rhodobacter capsularis, and Rhodopseudomonas sulfidophilus with a viable count of not less than 200 million / ml can be obtained by mixed culture, and then the photosynthetic bacterial composite flora is expanded and cultured for later use.

[0114] (3) The cultured photosynthetic bacterial composite flora was inoculated into 700 L of a culture solution of a bacterial agent having a pH value in the range of 7.7 to 7.8, wherein the formula of the culture solution of the bacterial agent is:

[0115] 160g magnesium sulfate, 1200g anhydrous sodium acetate, 35g anhydrous calcium chloride, 364g urea, 750g dipotassium hydrogen phosphate, 491.4g potassium dihydrogen phosphate, 100g yeast extract, 0.7g niacin, 7g boric acid and water sterilized by strong ultraviolet light. The culture barrel used is also sterilized by ozone, and the sterilized culture barrel is in an anaerobic environment and in a closed state through carbon dioxide or other deoxygenation.

[0116] After inoculating the cultured photosynthetic bacteria composite flora into the culture solution of the microbial agent under sterile conditions, the photosynthetic bacteria composite flora is light-cultured at a temperature of 33±3 for 20 hours to allow the photosynthetic bacteria composite flora to adapt to the environment of the culture barrel, and then 490g of sodium selenite is added at one time, and cultured for 720 hours under intermittent stirring speed, wherein the stirring interval is 3 minutes every 2 hours, to obtain a microbial agent, and the concentration of organic selenium in the microbial agent is measured to be 179.3mg / L, and the conversion rate of inorganic selenium is 84.6%.

[0117] Example 4

[0118] Compared with Example 1, the amount of sodium selenite added in step (3) of this comparative example is 300 g, and the remaining steps are the same as those in Example 1 to obtain a comparative bacterial agent. The concentration of organic selenium in the obtained bacterial agent can reach 118 mg / L, and the conversion rate of inorganic selenium is 91.5%.

[0119] Comparative Example 1

[0120] Compared with Example 1, this comparative example omits Rhodopseudomonas palustris in step (1), and the composite bacterial community obtained also does not contain Rhodopseudomonas palustris. The subsequent steps (2) and (3) are carried out using the composite bacterial community without Rhodopseudomonas palustris, and the remaining steps are the same as in Example 1 to obtain a comparative bacterial agent. The concentration of organic selenium in the bacterial agent is measured to be 153.6 mg / L, and the conversion rate of inorganic selenium is 72.5%.

[0121] Comparative Example 2

[0122] Compared with Example 1, in this comparative example, Rhodopseudomonas sulfophilus is omitted in step (1), and the composite bacterial community obtained also does not contain Rhodopseudomonas sulfophilus. The subsequent steps (2) and (3) are carried out using the composite bacterial community without Rhodopseudomonas sulfophilus, and the remaining steps are the same as in Example 1 to obtain a comparative bacterial agent. The concentration of organic selenium in the bacterial agent is measured to be 163.2 mg / L, and the conversion rate of inorganic selenium is 77%.

[0123] Comparative Example 3

[0124] Compared with Example 1, this comparative example omits Rhodospirilla crimson in step (1), and the composite bacterial community obtained also does not contain Rhodospirilla crimson. The subsequent steps (2) and (3) are carried out using the composite bacterial community without Rhodospirilla crimson, and the remaining steps are the same as in Example 1 to obtain a comparative bacterial agent. The concentration of organic selenium in the bacterial agent is measured to be 153.6 mg / L, and the conversion rate of inorganic selenium is 72.5%.

[0125] Comparative Example 4

[0126] Compared with Example 1, this comparative example omitted nitrogen-fixing red bacteria in step (1), and the composite bacterial community obtained also did not contain nitrogen-fixing red bacteria. The composite bacterial community without nitrogen-fixing red bacteria was used to carry out subsequent steps (2) and step (3), and the remaining steps were the same as in Example 1 to obtain a comparative bacterial agent. The concentration of organic selenium in the bacterial agent was measured to be 165.7 mg / L, and the conversion rate of inorganic selenium was 78.2%.

[0127] Comparative Example 5

[0128] Compared with Example 1, this comparative example omits the encapsulated red bacterium in step (1), and the obtained composite bacterial community also does not contain the encapsulated red bacterium. The subsequent steps (2) and (3) are carried out using the composite bacterial community without the encapsulated red bacterium. The remaining steps are the same as those in Example 1 to obtain a comparative bacterial agent. The concentration of organic selenium in the bacterial agent is measured to be 171.2 mg / L, and the conversion rate of inorganic selenium is 80.8%.

[0129] Comparative Example 6

[0130] Compared with Example 1, in step (3), the photosynthetic bacteria composite flora obtained by culture was inoculated into the culture solution of the microbial agent under aseptic conditions, and then the photosynthetic bacteria composite flora was cultured under light at a temperature of 20°C for 20 hours, and the remaining steps were the same as those in Example 1 to obtain a comparative microbial agent. The growth rate of the flora in the microbial agent was slow, and under the same growth time, the flora in the microbial agent did not obtain a visible number of viable bacteria (check the colony diameter and number).

[0131] Comparative Example 7

[0132] Compared with Example 1, in step (3), the photosynthetic bacteria composite flora obtained by culture was inoculated into the culture solution of the microbial agent under aseptic conditions, and then the photosynthetic bacteria composite flora was cultured under light at a temperature of 40°C for 20 hours, and the flora adhered to the wall seriously. The remaining steps were the same as those in Example 1 to obtain a comparative microbial agent. The concentration of organic selenium in the microbial agent was measured to be 130.5 mg / L, and the conversion rate of inorganic selenium was 61.5%.

[0133] It can be seen that the bacteria in the composite bacterial community provided in the present application synergistically promote the conversion of inorganic selenium, so that its conversion rate can reach more than 80%, and the composite bacterial community formed by it has good tolerance to inorganic selenium solutions and can grow in higher concentrations of inorganic selenium solutions, so that the content of organic selenium in the obtained bacterial agent is higher.

[0134] The total selenium content and the amount of organic selenium in the components of the bacterial agents obtained in Examples 1 to 4 and the bacterial agents obtained in Comparative Examples 1 to 7 were tested respectively, and the Beijing Qingxi Technology Research Institute was commissioned to detect the type and content of organic selenium in the bacterial agents (wherein the content of each selenium-containing organic matter in the organic selenium is the proportion of the selenium-containing organic matter in the total amount of organic selenium in mass percentage). The test results are shown in Table 8.

[0135] Table 8

[0136]

[0137] As can be seen from Table 8, the organic selenium in the bacterial agent formed by the conversion and treatment of inorganic selenium by the composite bacterial community provided by the present application is rich in types of organic selenium, and the contents of selenium polypeptides, selenoamino acids, and selenocysteine ​​that are easily absorbed by plants are significantly higher than those in the comparative example; and the conversion rate of inorganic selenium is also better. In addition, calcium protein and phospholipids also appear in the bacterial agent provided by the present application, which are not only beneficial to plant growth, but also for the absorption of organic selenium, especially for the organic selenium to be transported to the stems and leaves of plants after being absorbed, and for the increase of organic selenium in the stems and leaves of crops that are used for stem and leaf utilization. Among them, the sources of calcium protein and phospholipid may come from the metabolic formation of the composite bacterial community of this specific combination during the conversion of inorganic selenium.

[0138] In order to verify the effects of the types of organic selenium in the microbial agent, especially the contents of selenoamino acids, calcium proteins and phospholipids on the absorption, transformation and transfer of selenium in plants, the applicant conducted experiments on wheat and potato crops respectively, and prepared and diluted the microbial agents obtained in Examples 1 to 4 and Comparative Examples 1 to 7 with water to form a microbial agent solution of 10 g microbial agent / 1 L of water, and sprayed wheat, potato and wild rice stem three times in the early, middle and early harvesting stages of planting, with the same spraying amount, and used the crops not sprayed with the microbial agent solution as blank controls to compare the yield of the products and the selenium content and selenoamino acid content in the products. The results are shown in Table 9.

[0139] Table 9

[0140]

[0141]

[0142] In addition, the applicant also tested the selenium content in the stems, leaves and roots of potatoes, and the test results are shown in Table 10:

[0143] Table 10

[0144]

[0145] It can be seen that the organic selenium content in the microbial agent provided by the present application is high and rich in variety, which is more conducive to the increase in crop yield and selenium-enriched crops, and the organic selenium in the obtained selenium-enriched crops, especially the content of selenoamino acid organic selenium that is convenient for the human body to directly absorb and utilize, is significantly increased. In addition, the selenium in the microbial agent provided by the present application can be enriched in the stems, leaves, and roots of the crops obtained, so that the stems, leaves, and roots of the crops obtained are rich in organic selenium, so that the whole plant of the crops obtained can be used as a selenium-enriched crop, which is conducive to the utilization of the whole plant of crops, and the selenium-enriched microbial agent provided by the present application has a wider range of application and can be applied to mainly edible crops of different types.

[0146] The present invention can be implemented in other forms than those described above without departing from the spirit and scope of the present invention. The embodiments disclosed in the present application are examples and are not limited to these.

Claims

1. An organic selenium-rich bacterial agent, characterized in that: Including organic selenium obtained by converting inorganic selenium, the content of organic selenium is not less than 80% by mass fraction of the total selenium; The organic selenium includes selenoamino acids, selenopolypeptides, selenoproteins and selenopolysaccharides obtained by conversion of inorganic selenium; based on 100 parts by mass of organic selenium, the organic selenium includes 30 to 35 parts of selenopolypeptides, 18 to 25 parts of selenoproteins, 30 to 45 parts of selenopolysaccharides and 5 to 15 parts of selenoamino acids; the content of inorganic selenium in the organic selenium bacterial agent is not higher than 20% and not lower than 2% in terms of the mass fraction of the total selenium.

2. The organic selenium-rich bacterial agent according to claim 1, characterized in that: The selenoamino acid includes one or more of selenomethionine and selenocysteine.

3. The organic selenium-rich bacterial agent according to claim 1, characterized in that: The organic-selenium-rich bacterial agent also includes calcium protein and phospholipids. The concentration of the calcium protein in the organic-selenium-rich bacterial agent is not less than 0.001 g / L; the concentration of the phospholipid in the organic-selenium-rich bacterial agent is not less than 0.001 g / L.

4. The organic selenium-rich bacterial agent according to any one of claims 1 to 3, characterized in that: The organic selenium-rich bacterial agent is obtained by fermenting an inorganic selenium solution with a photosynthetic bacteria composite flora; wherein the photosynthetic bacteria composite flora includes Rhodopseudomonas palustris, Rhodospirillum rubrum, nitrogen-fixing Rhodobacter, Rhodobacter capsulatum, and Rhodopseudomonas thiophila.

5. The organic selenium-rich bacterial agent according to claim 4, characterized in that: The total viable count of the photosynthetic bacteria complex in the organic selenium-rich bacterial agent is not less than 200 million / mL.

6. A method for preparing an organic selenium-rich bacterial agent, characterized in that: The composite flora of photosynthetic bacteria is inoculated into a culture solution, and inorganic selenium salt is added to perform fermentation culture to obtain the product; The photosynthetic bacteria complex includes Rhodopseudomonas palustris, Rhodospirillum rubrum, Nitrogen-fixing Rhodobacter, Rhodobacter capsulatum, and Rhodopseudomonas thiophila.

7. The method for preparing the organic selenium-rich bacterial agent according to claim 6, characterized in that: The culture solution includes the following components by mass: 78-92 parts of magnesium sulfate, 720-880 parts of anhydrous sodium acetate, 32-38 parts of anhydrous calcium chloride, 450-550 parts of urea, 440-520 parts of dipotassium hydrogen phosphate, 280-340 parts of potassium dihydrogen phosphate, 95-115 parts of yeast extract, 22-28 parts of protein extract, 0.13-0.17 parts of niacin, 3.5-4.5 parts of boric acid and 570,000-700,000 parts of water; The concentration of the inorganic selenium salt formed by adding the inorganic selenium salt into the culture solution is 0.63-0.77 g / L.

8. The method for preparing the organic selenium-rich bacterial agent according to claim 7, characterized in that: The pH value of the culture solution is 7.7 to 7.8; and / or The fermentation culture conditions are: temperature 30-38, stirring speed 18-22r / min, stirring for 2.5-3.5min every 2.5-3.5 hours, and fermentation time 500-700h.

9. The method for preparing the organic selenium-rich bacterial agent according to claim 8, characterized in that: After the photosynthetic bacteria composite flora is inoculated into the culture solution, the culture is carried out for 18 to 22 hours, and then the inorganic selenium salt is added.

10. Use of the organic-selenium-rich bacterial agent prepared by the method for preparing the organic-selenium-rich bacterial agent according to any one of claims 1 to 5 or the organic-selenium-rich bacterial agent according to any one of claims 6 to 9 in crop planting.

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